Organic Anion Transporting Polypeptide 1B1 Research Articles

The Role of Adopted Orphan Nuclear Receptors in the Regulation of an Organic Anion Transporting Polypeptide 1B1 (OATP1B1) under the Action of Sex Hormones.

Organic anion transporting polypeptide 1B1 (OATP1B1) is an influx transporter protein of the SLC superfamily, expressed mainly in the liver and some tumor cells. The mechanisms of its regulation are being actively studied. In the present study, the effect of sex hormones (estradiol, progesterone and testosterone) on OATP1B1 expression in HepG2 cells was examined. The role of adopted orphan receptors, farnasoid X receptor (FXR), constitutive androstane receptor (CAR), pregnane X receptor (PXR) and liver X receptor subtype alpha (LXRa), was also evaluated. Hormones were used in concentrations of 1, 10 and 100 μM, with incubation for 24 h. The protein expression of OATP1B1, FXR, CAR, PXR and LXRa was analyzed by Western blot. It was shown that estradiol (10 and 100 μM) increased the expression of OATP1B1, acting through CAR. Testosterone (1, 10 and 100 μM) increased the expression of OATP1B1, acting through FXR, PXR and LXRa. Progesterone (10 and 100 μM) decreased the expression of OATP1B1 (10 and 100 μM) and adopted orphan receptors are not involved in this process. The obtained results have important practical significance and determine ways for targeted regulation of the transporter, in particular in cancer.

Near-Infrared Fluorescence Imaging of miRNA Using a Transmembrane Polypeptide-Based Genetic Reporter.

MicroRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in multiple biological processes. Many miRNAs exhibit unique expression patterns and are considered as theranostic biomarkers in a variety of human diseases. A reporter system that is capable of imaging miRNA in vivo is crucial for investigating miRNA biology. In the present study, an organic anion-transporting polypeptide 1B3 (OATP1B3)-based genetic switch system is designed and optimized to achieve near-infrared fluorescent imaging of miRNA by the uptake of indocyanine green (ICG) dye. The reporter system, named miR-ON-OB3, is shown to be efficient to regulate the expression of OATP1B3 in mammalian cells. Notably, the results indicate that the system is of high sensitivity for near-infrared fluorescence imaging of both exogenous and endogenous miRNA in mammalian cells. Moreover, the system is proved to be functional for real-time near-infrared fluorescence imaging of miRNA in living mice. This study establishes a novel genetic encoded reporter for near-infrared fluorescence imaging of miRNA, which may provide a potential tool for in vivo imaging of miRNA in clinical applications due to the clinical availability of ICG.

The Double-Leucine Motifs Affect Internalization, Stability, and Function of Organic Anion Transporting Polypeptide 1B1.

Organic anion transporting polypeptide 1B1 (OATP1B1) is specifically expressed at the basolateral membrane of human hepatocytes and plays important roles in the uptake of various endogenous and exogenous compounds including many drugs. The proper functioning of OATP1B1, hence, is essential for the bioavailability of various therapeutic agents and needs to be tightly regulated. Dileucine-based signals are involved in lysosomal targeting, internalization, and trans-Golgi network to endosome transporting of membrane proteins. In the current study, we analyzed the 3 intracellular and 13 transmembrane dileucine motifs (DLMs) within the sequence of OATP1B1. It was found that the simultaneous replacement of I332 and L333 with alanine resulted in a significantly reduced level of the mature form of OATP1B1. The cell surface expression of I332A/L333A could be partially rescued by MG132, as well as agents that prevent clathrin-dependent protein internalization, suggesting that this dileucine motif may be involved in the endocytosis of OATP1B1. On the other hand, I376/L377 and I642/L643, which are localized at transmembrane helices (TM) 8 and 12, respectively, are involved in the interaction of the transporter with its substrates. I642A/L643A exhibited a significantly decreased protein level compared to that of the wild-type, implying that the motif is important for maintaining the stability of OATP1B1 as well.

Importance of N-Glycosylation for the Expression and Function of Human Organic Anion Transporting Polypeptide 2B1.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a membrane transporter widely expressed in organs crucial for drug absorption and disposition such as the intestine, liver, and kidney. Evidence indicates that OATP2B1 is a glycoprotein. However, the sites of glycosylation and their contribution to the function and expression of OATP2B1 are largely unknown. In this study, by site-directed mutagenesis, we determined that two of four potential N-glycosylation sites in OATP2B1, N176 and N538, are indeed glycosylated. Functional studies revealed that the transport activities of mutants N176Q and N538Q were greatly reduced as compared to that of wild-type OATP2B1. However, the reduced activity was not due to the impairment of transport function per se but due to the decreased surface expression as the Km and normalized Vmax values of N176Q and N538Q were comparable to those of OATP2B1. Quantitative polymerase chain reaction (PCR) revealed that N176Q and N538Q mutations did not affect the expression of OATP2B1 at a transcriptional level. Immunofluorescence analysis showed that deglycosylated OATP2B1 was largely retained in the endoplasmic reticulum, which may activate the endoplasmic reticulum-associated degradation pathway, and the ubiquitin-proteasome system played a major role in the degradation of OATP2B1. Taken together, OATP2B1 is N-glycosylated, and N-glycosylation is essential for the surface expression of OATP2B1 but not critical for the transport function of OATP2B1 per se.

Utilization of Rosuvastatin and Endogenous Biomarkers in Evaluating the Impact of Ritlecitinib on BCRP, OATP1B1, and OAT3 Transporter Activity

PurposeRitlecitinib, an inhibitor of Janus kinase 3 and tyrosine kinase expressed in hepatocellular carcinoma family kinases, is in development for inflammatory diseases. This study assessed the impact of ritlecitinib on drug transporters using a probe drug and endogenous biomarkers.MethodsIn vitro transporter-mediated substrate uptake and inhibition by ritlecitinib and its major metabolite were evaluated. Subsequently, a clinical drug interaction study was conducted in 12 healthy adult participants to assess the effect of ritlecitinib on pharmacokinetics of rosuvastatin, a substrate of breast cancer resistance protein (BCRP), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporter 3 (OAT3). Plasma concentrations of coproporphyrin I (CP-I) and pyridoxic acid (PDA) were assessed as endogenous biomarkers for OATP1B1 and OAT1/3 function, respectively.ResultsIn vitro studies suggested that ritlecitinib can potentially inhibit BCRP, OATP1B1 and OAT1/3 based on regulatory cutoffs. In the subsequent clinical study, coadministration of ritlecitinib decreased rosuvastatin plasma exposure area under the curve from time 0 to infinity (AUCinf) by ~ 13% and maximum concentration (Cmax) by ~ 27% relative to rosuvastatin administered alone. Renal clearance was comparable in the absence and presence of ritlecitinib coadministration. PK parameters of AUCinf and Cmax for CP-I and PDA were also similar regardless of ritlecitinib coadministration.ConclusionRitlecitinib does not inhibit BCRP, OATP1B1, and OAT3 and is unlikely to cause a clinically relevant interaction through these transporters. Furthermore, our findings add to the body of evidence supporting the utility of CP-I and PDA as endogenous biomarkers for assessment of OATP1B1 and OAT1/3 transporter activity.

Predictive Modeling of HMG-CoA Reductase Inhibitory Activity and Design of New HMG-CoA Reductase Inhibitors.

As blood cholesterol increases, it accumulates in the intima of blood vessels, elevating the risk of atherosclerosis and coronary artery disease. Drugs that inhibit enzymes essential for cholesterol synthesis are effective in improving blood cholesterol levels. Statins are used to treat hypercholesterolemia as they inhibit 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), the rate-limiting enzyme in cholesterol synthesis. Statins are known to exert their effects by translocating to the liver, where they are taken up by the organic anion transporting polypeptide 1B1 (OATP1B1). Therefore, we hypothesized that a compound with high HMGR inhibitory activity and high affinity for OATP1B1 would be an excellent new therapeutic agent for hypercholesterolemia with increased liver selectivity and fewer side effects. In this study, we developed two models for predicting HMGR inhibitory activity and OATP1B1 affinity to propose the chemical structure of a new therapeutic agent for hypercholesterolemia with both high inhibitory activity and high liver selectivity. HMGR inhibitory activity and OATP1B1 affinity prediction models were constructed with high prediction accuracy for the test data: r2 = 0.772 and 0.768, respectively. New chemical structures were then input into these models to search for candidate compounds. We found compounds with higher HMGR inhibitory activity and OATP1B1 affinity than rosuvastatin, the most recently developed statin drug, and compounds that did not have a common structure of statins with high HMGR inhibitory activity.

Relevance of the organic anion transporting polypeptide 1B3 (OATP1B3) in the personalized pharmacological treatment of hepatocellular carcinoma

Although pharmacological treatment is the best option for most patients with advanced hepatocellular carcinoma (HCC), its success is very limited, partly due to reduced uptake and enhanced efflux of antitumor drugs. Here we have explored the usefulness of vectorizing drugs towards the organic anion transporting polypeptide 1B3 (OATP1B3) to enhance their efficacy against HCC cells. In silico studies (RNA-Seq data, 11 cohorts) and immunohistochemistry analyses revealed a marked interindividual variability, together with general downregulation but still expression of OATP1B3 in the plasma membrane of HCC cells. The measurement of mRNA variants in 20 HCC samples showed the almost absence of the cancer-type variant (Ct-OATP1B3) together with marked predominance of the liver-type variant (Lt-OATP1B3). In Lt-OATP1B3-expressing cells, the screening of 37 chemotherapeutical drugs and 17 tyrosine kinase receptors inhibitors (TKIs) revealed that 10 classical anticancer drugs and 12 TKIs were able to inhibit Lt-OATP1B3-mediated transport. Lt-OATP1B3-expressing cells were more sensitive than Mock parental cells (transduced with empty lentiviral vectors) to some Lt-OATP1B3 substrates (paclitaxel and the bile acid-cisplatin derivative Bamet-UD2), but not to cisplatin, which is not transported by Lt-OATP1B3. This enhanced response was abolished by competition with taurocholic acid, a known Lt-OATP1B3 substrate. Tumors subcutaneously generated in immunodeficient mice by Lt-OATP1B3-expressing HCC cells were more sensitive to Bamet-UD2 than those derived from Mock cells. In conclusion, Lt-OATP1B3 expression should be screened before deciding the use of anticancer drugs substrates of this carrier in the personalized treatment of HCC. Moreover, Lt-OATP1B3-mediated uptake must be considered when designing novel anti-HCC targeted drugs.

Cell-Dependent Activation of ProTide Prodrugs and Its Implications in Antiviral Studies.

The ProTide prodrug design is a powerful tool to improve cell permeability and enhance the intracellular activation of nucleotide antiviral analogues. Previous in vitro studies showed that the activation of ProTide prodrugs varied in different cell lines. In the present study, we investigated the activation profiles of two antiviral prodrugs tenofovir alafenamide (TAF) and sofosbuvir (SOF) in five cell lines commonly used in antiviral research, namely, Vero E6, Huh-7, Calu-3, A549, and Caco-2. We found that TAF and SOF were activated in a cell-dependent manner with Vero E6 being the least efficient and Huh-7 being the most efficient cell line for activating the prodrugs. We also demonstrated that TAF was activated at a significantly higher rate than SOF. We further analyzed the protein expressions of the activating enzymes carboxylesterase 1, cathepsin A, histidine triad nucleotide-binding protein 1, and the relevant drug transporters P-glycoprotein and organic anion-transporting polypeptides 1B1 and 1B3 in the cell lines using the proteomics data extracted from the literature and proteome database. The results revealed significant differences in the expression patterns of the enzymes and transporters among the cell lines, which might partially contribute to the observed cell-dependent activation of TAF and SOF. These findings highlight the variability of the abundance of activating enzymes and transporters between cell lines and emphasize the importance of selecting appropriate cell lines for assessing the antiviral efficacy of nucleoside/nucleotide prodrugs.

The Role of Coproporphyrins as Endogenous Biomarkers for Organic Anion Transporting Polypeptide 1B Inhibition - progress from 2016 to 2023.

Since the initial clinical study investigating CP-I and CP-III as endogenous biomarkers for OATP inhibition DDIs published in 2016, significant progress has been made in confirming the usefulness of the CPs, particularly the CP-I, as biomarkers in assessing OATP inhibition DDIs. CP-I exhibits selectivity towards OATP1B activity in human subjects with genetic variants of OATP1B1. Its sensitivity to a broad spectrum of clinical OATP1B inhibitors has been established, from weak to vigorous. Dose-dependent CP-I changes in healthy human subjects show agreement with DDI magnitudes of probe substrates by rifampin treatment. PBPK models have been established for concentration changes of plasma CP-I with OATP inhibitors, demonstrating the usefulness of supporting the quantitative translation of the effect of CP-I levels into the DDI risk assessment of potential OATP inhibitors. As plasma CP-I's sensitivity, specificity, and selectivity have been validated in humans, monitoring CP-I levels in single and multiple clinical phase I dose escalation studies is recommended for early assessment of DDI risks and understanding the full dose-response of an investigational drug to OATP inhibitions. A decision tree is proposed to preclude the need to conduct a dedicated DDI study by administering a probe substrate drug to human subjects. Significance Statement The mini-review summarized the validation paths of CP-I and CP-III as biomarkers of OATP1B inhibition in humans for their selectivity, specificity, and sensitivity. The utility of monitoring CP-I to assess drug-drug interactions of OATP1B inhibition in early drug development is proposed. Changes in plasma CP-I in phase I dose range studies can be used to frame plans for late-stage development and facilitate the mechanistic understanding of complex drug-drug interactions.

Potential Involvement of Organic Anion Transporters in Drug Interactions with Shuganning Injection, a Traditional Chinese Patent Medicine.

Traditional Chinese medicine injections have been widely used in China for the treatment of various diseases. Transporter-mediated drug-drug interactions are a major contributor to adverse drug reactions. However, the research on transporter-mediated Traditional Chinese medicine injection-drug interactions is limited. Shuganning injection is a widely used Traditional Chinese medicine injection for treating various liver diseases. In this study, we investigated the inhibitory effect of Shuganning injection and its four main ingredients (baicalin, geniposide, chlorogenic acid, and oroxylin A) on 9 drug transporters. Shuganning injection strongly inhibited organic anion transporter 1 and organic anion transporter 3 with IC50 values < 0.1% (v/v), and moderately inhibited organic anion transporter 2, organic anion transporting-polypeptide 1B1, and organic anion transporting-polypeptide 1B3 with IC50 values < 1.0%. Baicalin, the most abundant bioactive ingredient in the Shuganning injection, was identified as both an inhibitor and substrate of organic anion transporter 1, organic anion transporter 3, and organic anion transporting-polypeptide 1B3. Oroxylin A had the potential to act as both an inhibitor and substrate of organic anion transporting-polypeptide 1B1 and organic anion transporting-polypeptide 1B3. In contrast, geniposide and chlorogenic acid had no significant inhibitory effect on drug transporters. Notably, Shuganning injection markedly altered the pharmacokinetics of furosemide and atorvastatin in rats. Using Shuganning injection as an example, our findings support the implementation of transporter-mediated Traditional Chinese medicine injection-drug interactions in the development of Traditional Chinese medicine injection standards.

Effect of Chronic Hepatitis C on the Activity of the Membrane Transporters P-gp and OATP1B1/BCRP on Patients With Different Stages of Hepatic Fibrosis.

The activity of the membrane transporters organic anion-transporting polypeptide 1B1 (OATP1B1) & breast cancer resistance protein (BCRP) (rosuvastatin) and P-glycoprotein (P-gp) (fexofenadine) was evaluated in patients with chronic hepatitis C virus (HCV) infection (n = 28), genotypes 1 and 3, investigated before the treatment with direct-acting antiviral agents (Phase 1) and up to 30 days after the assessment of the virologic response (Phase 2). Participants allocated in Groups 1 (n = 15; F0/F1 and F2, mild to moderate liver fibrosis) and 2 (n = 13; F3 and F4, advanced course of liver fibrosis/cirrhosis) received in both phases fexofenadine (10 mg) and rosuvastatin (2 mg). OATP1B1 & BCRP activity (rosuvastatin area under the plasma concentration-time curve of rosuvastatin from time zero to infinity (AUC0-∞ )) was reduced in Groups 1 and 2, respectively, by 25% (ratio 0.75 (0.53-0.82), P < 0.01) and 31% (ratio 0.69 (0.46-0.85), P < 0.05) in Phase 1 compared with Phase 2. OATP1B1 & BCRP activity was reduced in Phases 1 and 2, respectively, by 49% (median ratio 1.51 (1.17-2.20), P < 0.05) and 61% (ratio 1.39 (1.16-2.02), P < 0.01) in Group 2 compared with Group 1. P-gp activity (fexofenadine AUC0-∞ ) was also reduced in Phase 1 compared with Phase 2 (ratio Phase2/Phase1 0.79 (0.66-0.96) in Group 1 and 0.81 (0.69-0.96) in Group 2) as well as in Group 2 compared with Group 1 in both Phases (ratio Group2/Group1 1.47 (1.08-2.01) in Phase 1 and 1.51 (1.10-2.07) in Phase 2). Thus, clinicians administering OATP1B1 & BCRP and P-gp substrates with low therapeutic indexes should consider the evolution of the treatment and the stage of HCV infection.

Characterization of Xenobiotic and Steroid Disposition Potential of Human Placental Tissue and Cell Lines (BeWo, JEG-3, JAR, and HTR-8/SVneo) by Quantitative Proteomics.

The placenta is a fetal organ that performs critical functions to maintain pregnancy and support fetal development, including metabolism and transport of xenobiotics and steroids between the maternal-fetal unit. In vitro placenta models are used to study xenobiotic and steroid disposition, but how well these models recapitulate the human placenta is not well understood. We first characterized the abundance of proteins involved in xenobiotic and steroid disposition in human placental tissue. In pooled human placenta, the following xenobiotic and steroid disposition proteins were detected (highest to lowest), 1) enzymes: glutathione S-transferase P, carbonyl reductase 1, aldo-keto reductase 1B1, hydroxysteroid dehydrogenases (HSD3B1 and HSD11B1), aromatase, epoxide hydrolase 1 (EPHX1) and steryl-sulfatase, and 2) transporters: monocarboxylate transporters (MCT1 and 4), organic anion transporting polypeptide 2B1, organic anion transporter 4, and breast cancer resistance protein (BCRP). Then, the tissue proteomics data were compared with four placental cell lines (BeWo, JEG-3, JAR, and HTR-8/SVneo). The differential global proteomics analysis revealed that the tissue and cell lines shared 1420 cytosolic and 1186 membrane proteins. Although extravillous trophoblast and cytotrophoblast marker proteins were detected in all cell lines, only BeWo and JEG-3 cells expressed the syncytiotrophoblast marker, chorionic somatomammotropin hormone 1. BeWo and JEG-3 cells expressed most target proteins including aromatase, HSDs, EPHX1, MCT1, and BCRP. JEG-3 cells treated with commonly detected phthalates in human biofluids showed dysregulation of steroid pathways. The data presented here show that BeWo and JEG-3 cells are closer to the placental tissue for studying xenobiotic and steroid disposition. SIGNIFICANCE STATEMENT: This is the first study to compare proteomics data of human placental tissue and cell lines (BeWo, JAR, JEG-3, and HTR-8/SVneo). The placental cell line and tissue proteomes are vastly different, but BeWo and JEG-3 cells showed greater resemblance to the tissue in the expression of xenobiotic and steroid disposition proteins. These data will assist researchers to select an optimum cell model for mechanistic investigations on xenobiotic and steroid disposition in the placenta.

Evaluation of the selectivity of several OATP1B biomarkers using relative activity factor method.

In recent years, some endogenous substrates of organic anion transporting polypeptide 1B (OATP1B) have been identified and characterized as potential biomarkers to assess OATP1B-mediated clinical drug-drug interactions (DDIs). However, quantitative determination of their selectivity to OATP1B are still limited. In this study, we developed a relative activity factor (RAF) method to determine the relative contribution of hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1, and sodium-taurocholate co-transporting polypeptide (NTCP) on hepatic uptake of several OATP1B biomarkers, including coproporphyrins I (CPI), CPIII, sulfate conjugates of bile acids: glycochenodeoxycholic acid sulfate (GCDCA-S), glycodeoxycholic acid sulfate (GDCA-S), and taurochenodeoxycholic acid sulfate (TCDCA-S). RAF values for OATP1B1, OATP1B3, OATP2B1, and NTCP were determined in cryopreserved human hepatocytes and transporter transfected cells using pitavastatin, cholecystokinin, resveratrol-3-O-β-D-glucuronide, and taurocholic acid (TCA) as reference compounds, respectively. OATP1B1-specific pitavastatin uptake in hepatocytes was measured in the absence and presence of 1 µM estropipate, while NTCP-specific TCA uptake was measured in the presence of 10 µM rifampin. Our studies suggested that CPI was a more selective biomarker for OATP1B1 than CPIII, while GCDCA-S and TCDCA-S were more selective to OATP1B3. OATP1B1 and OATP1B3 equally contributed to hepatic uptake of GDCA-S. The mechanistic static model, incorporating the fraction transported (ft) of CPI/III estimated by RAF and in vivo elimination data, predicted several perpetrator interactions with CPI/III. Overall, RAF method combined with pharmacogenomic and DDI studies is a useful tool to determine the selectivity of transporter biomarkers and facilitate the selection of appropriate biomarkers for DDI evaluation. Significance Statement We developed a new RAF method to quantitatively determine the contribution of hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1, and NTCP on several OATP1B biomarkers (CPI, CPIII, GCDCA-S, GDCA-S, and TCDCA-S) and evaluated their predictivity on perpetrator-biomarker interactions. Our studies suggest that RAF method is a useful tool to determine the selectivity of transporter biomarkers. This method combined with pharmacogenomic and DDI studies will facilitate mechanistic interpretation and modeling of biomarker data and the selection of appropriate biomarkers for DDI evaluation.

Developing a mechanistic understanding of the nonlinear pharmacokinetics of letermovir and prospective drug interaction with everolimus using physiological-based pharmacokinetic modeling.

Letermovir is approved for use in cytomegalovirus-seropositive hematopoietic stem cell transplant recipients and is investigated in other transplant settings. Nonlinear pharmacokinetics (PKs) were observed in clinical studies after intravenous and oral dosing across a wide dose range, including the efficacious doses of 240 and 480 mg. A physiologically-based PK (PBPK) model for letermovir was built to develop a plausible explanation for the nonlinear PKs observed in clinical studies. In vitro studies suggested that letermovir elimination and distribution are mediated by saturable uridine glucuronosyltransferases (UGT)-metabolism and by saturable hepatic uptake via organic anion-transporting polypeptides (OATP) 1B. A sensitivity analysis of parameters describing the metabolism and distribution mechanisms indicated that the greater than dose-proportional increase in letermovir exposure is best described by a saturable OATP1B-mediated transport. This PBPK model was further used to evaluate the drug interaction potential between letermovir and everolimus, an immunosuppressant that may be co-administered with letermovir depending on regions. Because letermovir inhibits cytochrome P450 (CYP) 3A and everolimus is a known CYP3A substrate, an interaction when concomitantly administered is anticipated. The drug-drug interaction simulation confirmed that letermovir will likely increase everolimus are under the curve by 2.5-fold, consistent with the moderate increase in exposure observed with midazolam in the clinic. The output highlights the importance of drug monitoring, which is common clinical practice for everolimus to maintain safe and efficacious drug concentrations in the targeted patient population when concomitantly administered with letermovir.

Evaluation of the drug-drug interaction potential of the novel hepatitis B and D virus entry inhibitor bulevirtide at OATP1B in healthy volunteers.

Introduction: Bulevirtide is a first-in-class antiviral drug to treat chronic hepatitis B/D. We investigated the drug-drug interaction potential and pharmacokinetics of high-dose subcutaneous bulevirtide (5mg twice daily) with organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (CYP) 3A4. Methods: This was a single-center, open-label, fixed-sequence drug-drug interaction trial in 19 healthy volunteers. Before and at bulevirtide steady state, participants ingested a single 40mg dose of pravastatin. A midazolam microdose was applied to quantify CYP3A4 activity. Results: At bulevirtide steady state, pravastatin area under the concentration-time curve (AUC0-∞) increased 1.32-fold (90% CI 1.08-1.61). The 5mg bulevirtide twice-daily treatment resulted in a mean AUC0-12 of 1210h*ng/ml (95% CI 1040-1408) and remained essentially unchanged under the influence of pravastatin. CYP3A4 activity did not change to a clinically relevant extent. As expected, total bile acids increased substantially (35-fold) compared to baseline during bulevirtide treatment. All study medication was well tolerated. Discussion: The study demonstrated that high-dose bulevirtide inhibited OATP1B-mediated hepatic uptake of the marker substrate pravastatin but the extent is considered clinically not relevant. Changes in CYP3A4 activity were also not clinically relevant. In conclusion, this study suggests that OATP1B substrate drugs as well as CYP3A4 substrates may safely be used without dose adjustment in patients treated with bulevirtide. However, in patients using high statin doses and where concomitant factors potentially further increase statin exposure, caution may be required when using bulevirtide.

Organic anion-transporting polypeptide 2B1 knockout and humanized mice; insights into the handling of bilirubin and drugs

Organic anion transporting polypeptide 2B1 (OATP2B1/SLCO2B1) facilitates uptake transport of structurally diverse endogenous and exogenous compounds. To investigate the roles of OATP2B1 in physiology and pharmacology, we established and characterized Oatp2b1 knockout (single Slco2b1-/- and combination Slco1a/1b/2b1-/-) and humanized hepatic and intestinal OATP2B1 transgenic mouse models. While viable and fertile, these strains exhibited a modestly increased body weight. In males, unconjugated bilirubin levels were markedly reduced in Slco2b1-/- compared to wild-type mice, whereas bilirubin monoglucuronide levels were modestly increased in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice. Single Slco2b1-/- mice showed no significant changes in oral pharmacokinetics of several tested drugs. However, markedly higher or lower plasma exposure of pravastatin and the erlotinib metabolite OSI-420, respectively, were found in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice, while oral rosuvastatin and fluvastatin behaved similarly between the strains. In males, humanized OATP2B1 strains showed lower conjugated and unconjugated bilirubin levels than control Slco1a/1b/2b1-deficient mice. Moreover, hepatic expression of human OATP2B1 partially or completely rescued the impaired hepatic uptake of OSI-420, rosuvastatin, pravastatin, and fluvastatin in Slco1a/1b/2b1-/- mice, establishing an important role in hepatic uptake. Expression of human OATP2B1 in the intestine was basolateral and markedly reduced the oral availability of rosuvastatin and pravastatin, but not of OSI-420 and fluvastatin. Neither lack of Oatp2b1, nor overexpression of human OATP2B1 had any effect on fexofenadine oral pharmacokinetics. While these mouse models still have limitations for human translation, with additional work we expect they will provide powerful tools to further understand the physiological and pharmacological roles of OATP2B1.

Visualizing cell–cell communication using synthetic notch activated MRI

Cell-cell communication plays a fundamental role in multicellular organisms. Cell-based cancer immunotherapies rely on the ability of innate or engineered receptors on immune cells to engage specific antigens on cancer cells to induce tumor kill. Toimprove the development and translation of these therapies, imaging tools capable of noninvasively and spatiotemporally visualizing immune-cancer cell interactions would be highly valuable. Using the synthetic Notch (SynNotch) system, we engineered T cells that upon interaction with a chosen antigen (CD19) on neighboring cancer cells induce the expression of optical reporter genes and the human-derived, magnetic resonance imaging (MRI) reporter gene organic anion transporting polypeptide 1B3 (OATP1B3). Administration of engineered T cells induced the antigen-dependent expression of all our reporter genes in mice bearing CD19-positive tumors but not CD19-negative tumors. Notably, due to the high spatial resolution and tomographic nature of MRI, contrast-enhanced foci within CD19-positive tumors representing OATP1B3-expressing T cells were clearly visible and their distribution was readily mapped. We then extended this technology onto human natural killer-92 (NK-92) cells, observing similar CD19-dependent reporter activity in tumor-bearing mice. Furthermore, we show that when delivered intravenously, engineered NK-92 cells can be detected via bioluminescence imaging in a systemic cancer model. With continued work, this highly modular imaging strategy could aid in the monitoring of cell therapies in patients and, beyond this, augment our understanding of how different cell populations interact within the body during normal physiology or disease.

Functional Characterization of Six SLCO1B1 (OATP1B1) Variants Observed in Finnish Individuals with a Psychotic Disorder.

Variants in the SLCO1B1 (solute carrier organic anion transporter family member 1B1) gene encoding the OATP1B1 (organic anion transporting polypeptide 1B1) protein are associated with altered transporter function that can predispose patients to adverse drug effects with statin treatment. We explored the effect of six rare SLCO1B1 single nucleotide variants (SNVs) occurring in Finnish individuals with a psychotic disorder on expression and functionality of the OATP1B1 protein. The SUPER-Finland study has performed exome sequencing on 9381 individuals with at least one psychotic episode during their lifetime. SLCO1B1 SNVs were annotated with PHRED-scaled combined annotation-dependent (CADD) scores and the Ensembl variant effect predictor. In vitro functionality studies were conducted for the SNVs with a PHRED-scaled CADD score of >10 and predicted to be missense. To estimate possible changes in transport activity caused by the variants, transport of 2',7'-dichlorofluorescein (DCF) in OATP1B1-expressing HEK293 cells was measured. According to the findings, additional tests with rosuvastatin and estrone sulfate were conducted. The amount of OATP1B1 in crude membrane fractions was quantified using a liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomics analysis. Six rare missense variants of SLCO1B1 were identified in the study population, located in transmembrane helix 3: c.317T>C (p.106I>T), intracellular loop 2: c.629G>T (p.210G>V), c.633A>G (p.211I>M), c.639T>A (p.213N>L), transmembrane helix 6: 820A>G (p.274I>V), and the C-terminal end: 2005A>C (p.669N>H). Of these variants, SLCO1B1 c.629G>T (p.210G>V) resulted in the loss of in vitro function, abolishing the uptake of DCF, estrone sulfate, and rosuvastatin and reducing the membrane protein expression to 31% of reference OATP1B1. Of the six rare missense variants, SLCO1B1 c.629G>T (p.210G>V) causes a loss of function of OATP1B1 transport in vitro and severely decreases membrane protein abundance. Carriers of SLCO1B1 c.629G>T might be susceptible to altered pharmacokinetics of OATP1B1 substrate drugs and might have increased likelihood of adverse drug effects such as statin-associated musculoskeletal symptoms.

Development of predictive QSAR models for the substrates/inhibitors of OATP1B1 by deep neural networks

The organic anion transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter. Inhibition of its normal function could lead to drug-drug interactions. In silico prediction is an effective means to identify potential OATP1B1 inhibitors and quantitative structure-activity relationship (QSAR) modeling is extensively used. As the structures of OATP1B1 substrates/inhibitors are quite diverse, machine learning based methods should be a good option for their QSAR analysis. In the present study, deep neural networks (DNNs) were employed to develop QSAR models for the substrates/inhibitors of OATP1B1 with different molecular fingerprints. Our results showed that QSAR models based on 4-hidden layer DNNs and ECFP4/FCFP4 fingerprints had the best generalization performance. The correlation coefficients (R2) of test set for ECFP4 and FCFP4 models were 0.641 and 0.653, respectively. Model application domain (AD) was calculated with Euclidean distance-based method, and AD could improve the performance of ECFP4 model but has little effect on FCFP4 model. Finally, the prediction of additional 8 compounds that not included in the data set further demonstrated that our QSAR models had a good predictive ability (averaged prediction accuracy >92%). The developed QSAR models could be used to screen large data sets and discover novel inhibitors for OATP1B1.

A single high-dose irradiation changes accumulation of methotrexate and gene expression levels of SLC and ABC transporters in cancer cells.

Chemoradiotherapy is frequently used to treat cancer. Stereotactic body radiotherapy (SBRT) is a single high-dose radiotherapy used to treat a variety of cancers. The anticancer drug methotrexate (MTX) shows affinity for solute carrier (SLC) and ATP-binding cassette (ABC) transporters. This study investigated relationships between accumulation of methotrexate and gene expression levels of solute carrier and ATP-binding cassette transporters in cancer cells after a single and high-dose X-ray irradiation. Cancer cell lines were selected from lung and cervical cancer cell line that are commonly used for stereotactic body radiotherapy and effective with methotrexate. We examined expression levels of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, OATP1B7, and organic anion transporter (OAT)1 as solute carrier transporters and multidrug resistance-associated protein (MRP)1 and MRP2 as ATP-binding cassette transporters, using real-time polymerase chain reaction and accumulation of 3H-MTX in cancer cells after 10-Gy irradiation, assuming stereotactic body radiotherapy. Cells were divided into three groups: Control without irradiation; 4h after irradiation; and 24h after irradiation. In control, gene expression levels of OAT1 in all cells was below the limit of measurement. After irradiation, gene expression levels of OATP1B1/1B3/1B7 showed changes in each cell line. Gene expression levels of MRP1/2 tended to increase after irradiation. Gene expression levels of OATP1B1/1B3/1B7 were much lower than those of MRP1/2. Accumulation of 3H-MTX tended to decrease over time after irradiation. Irradiation of cancer cells thus alters gene expression levels of both solute carrier transporters (OATP1B1/1B3/1B7) and ABC transporters (MRP1/2) and decreases accumulation of 3H-MTX in cancer cells over time due to elevated expression of MRP1/2.